The scrapie prion protein (PrPSc) is the major component of the infectious particle (prion) responsible for a group of fatal neurodegenerative disorders, including Creutzfeldt-Jakob disease and kuru in human beings, and scrapie in animals. During prion infection, PrpSc is generated by posttranslational modification of a glycolipid-anchored membrane protein of the host called PrPC. While PrpSc has been the subject of intensive study, much less attention has been paid to PrPC, whose physiological function is unknown. The purpose of the present application is to explore several interrelated questions concerning the cellular properties of PrPC, with a view to understanding the normal function of this isoform, as well as to establishing possible therapeutic strategies for inhibiting its conversion to PrPSC. The proposed studies are relevant to Alzheimer's disease as well as to prion disease, since both disorders are dementing amyloidoses that are likely to result from aberrant cellular processing of a membrane-anchored precursor protein. First, we propose to characterize, purify, and molecularly clone a cell surface protein that we hypothesize serves as a receptor for membrane- anchored and soluble forms of PrPC, and that mediates endocytic uptake of these forms via clathrin-coated pits. Using PrP bacterial fusion proteins as ligands, we will perform binding assays to quantitate the number and affinity of the receptors, chemical cross-linking to define their molecular properties, and conventional as well as affinity chromatography to purify them for microsequencing. Second, we propose to analyze the trafficking of prPC in polarized cell types, including cultured hippocampal neurons, PC12 cells, and MDCK cells. We will use immunofluorescence staining and biochemical techniques to determine whether PrPC, like several other glycolipid-anchored proteins, is preferentially localized to axonal and apical surfaces; whether it cycles through axonal or somato-dendritic endosomes; and whether it is present in synaptic vesicles. Third, since recent evidence indicates that prion synthesis occurs along an endocytic pathway, we plan to identify agents which act as inhibitors of PrPC endocytosis, and then test their effects on PrPSc synthesis in scrapie-infected neuroblastoma cells. We will test PrP peptides and bacterial fusion proteins that competitively inhibit binding of cell surface PrPC to its putative receptor, as well as anti-PrP antibodies, and compounds that disrupt clathrin lattices.